The goal of this project is to identify molecular characteristics of multi-drug resistance in high-risk neuroblastoma patients with single-cell technology. We propose a paradigm-shift approach to map mutations into a single cell and identify sub-clones by molecular similarity of individual cells. Neuroblastoma is the common solid tumor in children, and it accounts for 15% of deaths from cancer in children. Here, we propose to identify multidrug-resistant mutations with Next Generation Sequencing (NGS) based on mutation frequency changes (from minority to majority) due to treatment selection. These NGS-identified mutations will be localized into individual cells with targeted single-cell RNA-seq to show that these mutations exist in a clone (a group of single-cells) and the clones expand during treatment. Finally, the resistant sub-clones will be isolated or generated for functional assays and distributed to research community. This approach shifts the paradigm of tumor molecular characterization by clustering molecularly similar cells into a clone rather than isolating a clone (often with experimental bias) for characterization. The results have direct and immediate impacts on current treatment of neuroblastoma. The molecular profiles of resistant clones can be developed into sensitive PCR assays for early detection of drug-resistance at diagnosis, and for personalized therapies to overcome treatment resistance at specific patients (who have specific sub-clones during chemotherapy). The single-cell approach that we develop in this proposal would be directly relevant to studies of other childhood and adult cancers as well.